Arginase Inhibition Mitigates Bortezomib-Exacerbated Cardiotoxicity in Multiple Myeloma.

BACKGROUND: Multiple myeloma (MM) is associated with increased cardiovascular morbidity and mortality, while MM therapies also result in adverse cardiac effects. Endothelial dysfunction and impaired nitric oxide (NO) pathway is their possible mediator. OBJECTIVE: Since MM is associated with increased arginase expression, resulting in the consumption of ʟ-arginine, precursor for NO synthesis, our aim was to test if cardiotoxicity mediated by MM and MM therapeutic, bortezomib (a proteasome inhibitor), can be ameliorated by an arginase inhibitor through improved endothelial function. METHODS: We used a mouse Vĸ*MYC model of non-light chain MM. Cardiac function was assessed by echocardiography. RESULTS: MM resulted in progressive left ventricular (LV) systolic dysfunction, and bortezomib exacerbated this effect, leading to significant impairment of LV performance. An arginase inhibitor, OAT-1746, protected the heart against bortezomib- or MM-induced toxicity but did not completely prevent the effects of the MM+bortezomib combination. MM was associated with improved endothelial function (assessed as NO production) vs. healthy controls, while bortezomib did not affect it. OAT-1746 improved endothelial function only in healthy mice. NO plasma concentration was increased by OAT-1746 but was not affected by MM or bortezomib. CONCLUSIONS: Bortezomib exacerbates MM-mediated LV systolic dysfunction in a mouse model of MM, while an arginase inhibitor partially prevents it. Endothelium does not mediate either these adverse or beneficial effects. This suggests that proteasome inhibitors should be used with caution in patients with advanced myeloma, where the summation of cardiotoxicity could be expected. Therapies aimed at the NO pathway, in particular arginase inhibitors, could offer promise in the prevention/treatment of cardiotoxicity in MM.

Immune checkpoint inhibition improves antimyeloma activity of bortezomib and STING agonist combination in Vk*MYC preclinical model.

Multiple myeloma (MM), a hematological malignancy of plasma cells, has remained incurable despite the development of novel therapies that improve patients' outcome. Recent evidence indicates that the stimulator of interferon genes (STING) pathway may represent a novel target for induction of antitumor immune response in multiple myeloma. Here, we investigated antitumor effects of STING agonist with bortezomib with or without checkpoint inhibitor in the treatment of MM. METHODS: STING expression in bone marrow plasma cells of 58 MM patients was examined by immunohistochemical staining. The effectiveness of the proposed therapy was evaluated in vivo in a syngeneic transplantable mouse model of MM (Vĸ*MYC) in immunocompetent mice. Flow cytometry was used to assess tumor burden and investigate activation of immune response against MM. ELISA was performed to measure serum inflammatory cytokines concentrations upon treatment. RESULTS: Combining a STING agonist [2'3'-cGAM(PS)2] with bortezomib significantly decreased tumor burden and improved the survival of treated mice compared to either of the compounds used alone. The combination treatment led to secretion of pro-inflammatory cytokines and increased the percentage of neutrophils, activated dendritic cells and T cells in the tumor microenvironment. However, it resulted also in increased expression of PD-L1 on the surface of the immune cells. Addition of anti-PD1 antibody further potentiated the therapeutic effects. CONCLUSIONS: Our findings indicate high antimyeloma efficacy of the three-drug regimen comprising bortezomib, STING agonist, and a checkpoint inhibitor.

Targeting arginase-1 exerts antitumor effects in multiple myeloma and mitigates bortezomib-induced cardiotoxicity.

Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here, we investigated the role of arginase 1 (ARG1) in Vκ*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in ʟ-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of Vκ*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However, arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether, these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors.

Potent but transient immunosuppression of T-cells is a general feature of CD71+ erythroid cells.

CD71+ erythroid cells (CECs) have been recently recognized in both neonates and cancer patients as potent immunoregulatory cells. Here, we show that in mice early-stage CECs expand in anemia, have high levels of arginase 2 (ARG2) and reactive oxygen species (ROS). In the spleens of anemic mice, CECs expansion-induced L-arginine depletion suppresses T-cell responses. In humans with anemia, CECs expand and express ARG1 and ARG2 that suppress T-cells IFN-γ production. Moreover, bone marrow CECs from healthy human donors suppress T-cells proliferation. CECs differentiated from peripheral blood mononuclear cells potently suppress T-cell activation, proliferation, and IFN-γ production in an ARG- and ROS-dependent manner. These effects are the most prominent for early-stage CECs (CD71highCD235adim cells). The suppressive properties disappear during erythroid differentiation as more differentiated CECs and mature erythrocytes lack significant immunoregulatory properties. Our studies provide a novel insight into the role of CECs in the immune response regulation.

Inhibition of arginase modulates T-cell response in the tumor microenvironment of lung carcinoma.

Immunotherapy has demonstrated significant activity in a broad range of cancer types, but still the majority of patients receiving it do not maintain durable therapeutic responses. Amino acid metabolism has been proposed to be involved in the regulation of immune response. Here, we investigated in detail the role of arginase 1 (Arg1) in the modulation of antitumor immune response against poorly immunogenic Lewis lung carcinoma. We observed that tumor progression is associated with an incremental increase in the number of Arg1+ myeloid cells that accumulate in the tumor microenvironment and cause systemic depletion of ʟ-arginine. In advanced tumors, the systemic concentrations of ʟ-arginine are decreased to levels that impair the proliferation of antigen-specific T-cells. Systemic or myeloid-specific Arg1 deletion improves antigen-induced proliferation of adoptively transferred T-cells and leads to inhibition of tumor growth. Arginase inhibitor was demonstrated to modestly inhibit tumor growth when used alone, and to potentiate antitumor effects of anti-PD-1 monoclonal antibodies and STING agonist. The effectiveness of the combination immunotherapy was insufficient to induce complete antitumor responses, but was significantly better than treatment with the checkpoint inhibitor alone. Together, these results indicate that arginase inhibition alone is of modest therapeutic benefit in poorly immunogenic tumors; however, in combination with other treatment strategies it may significantly improve survival outcomes.

Automated semi-real-time detection of muscle activity with ultrasound imaging.

Ultrasound imaging (USI) biofeedback is a useful therapeutic tool; however, it relies on qualitative assessment by a trained therapist, while existing automatic analysis techniques are computationally demanding. This study aims to present a computationally inexpensive algorithm based on the difference in pixel intensity between USI frames. During an offline experiment, where data was analyzed after the study, participants performed isometric contractions of the gastrocnemius medialis (GM) muscle, as executed (30% of maximum contraction) or attempted (low force contraction up to a point when the participant is aware of exerting force or contracting the muscle) movements, while USI, EMG, and force data were recorded. The algorithm achieved 99% agreement with EMG and force measurements for executed movements and 93% for attempted movements, with USI detecting 1.9% more contractions than the other methods. In the online study, participants performed GM muscle contractions at 10% and 30% of maximum contraction, while the algorithm provided visual feedback proportional to the muscle activity (based on USI recordings during the maximum contraction) in less than 3 s following each contraction. We show that the participants reached the target consistently, learning to perform precise contractions. The algorithm is reliable and computationally very efficient, allowing real-time applications on standard computing hardware. It is a suitable method for automated detection, quantification of muscle contraction, and to provide biofeedback which can be used for training of targeted muscles, making it suitable for rehabilitation. Biofeedback session based on ultrasound imaging (USI) during muscle training. Novel, computationally inexpensive algorithm based on the difference in pixel intensity between USI frames is used to process the video and provide quantitative feedback on the strength of muscle contraction.

Mosaic IL6ST variant inducing constitutive GP130 cytokine receptor signaling as a cause of neonatal onset immunodeficiency with autoinflammation and dysmorphy.

Interleukin-6 signal transducer (IL6ST) encodes the GP130 protein which transduces the proinflammatory signaling of the IL6 cytokine family through Janus kinase signal transducers and activators of transcription pathway (JAK/STAT) activation. Biallelic loss-of-function IL6ST variants cause autosomal recessive hyper-IgE syndrome or a variant of the Stuve-Wiedemann syndrome. Somatic gain-of-function IL6ST mutations, in particular, small monoallelic in-frame deletions of which the most prevalent is the IL6ST Ser187_Tyr190del, are an established cause of inflammatory hepatocellular tumors, but so far, no disease caused by such mutations present constitutively has been described. Herein, we report a pediatric proband with a novel syndrome of neonatal onset immunodeficiency with autoinflammation and dysmorphy associated with the IL6ST Tyr186_Tyr190del variant present constitutively. Tyr186_Tyr190del was found by exome sequencing and was shown to be de novo (absent in proband's parents and siblings) and mosaic (present in approximately 15-40% of cells depending on the tissue studied-blood, urine sediment, hair bulbs and buccal swab). Functional studies were performed in the Epstein-Barr virus-immortalized patient's B cell lymphoblastoid cell line, which carried the variant in approximately 95% of the cells. Western blot showed that the patient's cells exhibited constitutive hyperphosphorylation of Tyr705 in STAT3, which is indicative of IL6-independent activation of GP130. Interestingly, the STAT3 phosphorylation could be inhibited with ruxolitinib as well as tofacitinib, which are clinically approved JAK1 and JAK3 (to lesser extent JAK2 and JAK1) inhibitors, respectively. Given our results and the recent reports of ruxolitinib and tofacitinib use for the treatment of diseases caused by direct activation of STAT3 or STAT1, we speculate that these drugs may be effective in the treatment of our patient's condition.

Myeloid Cell-Derived Arginase in Cancer Immune Response.

Amino acid metabolism is a critical regulator of the immune response, and its modulating becomes a promising approach in various forms of immunotherapy. Insufficient concentrations of essential amino acids restrict T-cells activation and proliferation. However, only arginases, that degrade L-arginine, as well as enzymes that hydrolyze L-tryptophan are substantially increased in cancer. Two arginase isoforms, ARG1 and ARG2, have been found to be present in tumors and their increased activity usually correlates with more advanced disease and worse clinical prognosis. Nearly all types of myeloid cells were reported to produce arginases and the increased numbers of various populations of myeloid-derived suppressor cells and macrophages correlate with inferior clinical outcomes of cancer patients. Here, we describe the role of arginases produced by myeloid cells in regulating various populations of immune cells, discuss molecular mechanisms of immunoregulatory processes involving L-arginine metabolism and outline therapeutic approaches to mitigate the negative effects of arginases on antitumor immune response. Development of potent arginase inhibitors, with improved pharmacokinetic properties, may lead to the elaboration of novel therapeutic strategies based on targeting immunoregulatory pathways controlled by L-arginine degradation.

Tissue clearing-based method for unobstructed three-dimensional imaging of mouse penis with subcellular resolution.

Although mice are widely used to elucidate factors contributing to penile disorders and develop treatment options, quantification of tissue changes upon intervention is either limited to minuscule tissue volume (histology) or acquired with limited spatial resolution (MRI/CT). Thus, imaging method suitable for expeditious acquisition of the entire mouse penis with subcellular resolution is described that relies on both aqueous- (clear, unobstructed brain imaging cocktails and computational analysis) and solvent-based (fluorescence-preserving capability imaging of solvent-cleared organs) tissue optical clearing (TOC). The combined TOC approach allows to image mouse penis innervation and vasculature with unprecedented detail and, for the first time, reveals the three-dimensional structure of murine penis fibrocartilage.

Systematic Evaluation of Chemically Distinct Tissue Optical Clearing Techniques in Murine Lymph Nodes.

Activation of adaptive immunity is a complex process coordinated at multiple levels in both time and the three-dimensional context of reactive lymph nodes (LNs). Although microscopy-based visualization of its spatiotemporal dynamics unravels complexities of developing immune response, such approach is highly limited by light-obstructing nature of tissue components. Recently, tissue optical clearing (TOC) techniques were established to bypass this obstacle and now allow to image and quantify the entire murine organs with cellular resolution. However, the spectrum of TOC is represented by wide variety of chemically distinct methods, each having certain advantages and disadvantages that were unsatisfactorily compared for suitability to LNs clearing. In this study, we have systematically tested 13 typical TOC techniques and assessed their impact on a number of critical factors such as LN transparency, imaging depth, change in size, compatibility with proteinaceous fluorophores, immunostaining, H&E staining, and light-sheet fluorescence microscopy. Based on the detailed data specific to TOC process of murine LNs, we provide a reliable reference for most suitable methods in an application-dependent manner.

Extracellular vesicles released by ovarian carcinoma contain arginase 1 that mitigates antitumor immune response.

Expression of arginase-1 (ARG1) is an immunosuppressive feature of tumor microenvironment that leads to depletion of ʟ-arginine, a nutrient required for T-cells expansion. Ovarian carcinoma cells release extracellular vesicles carrying enzymatically active ARG1, that contributes to local and systemic immune suppression, which can be restored by ARG inhibitor.

Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma.

Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule - ARG1, mitigating anti-tumor immune responses.

Cortical Functional Domains Show Distinctive Oscillatory Dynamic in Bimanual and Mirror Visual Feedback Tasks.

It is believed that Mirror Visual Feedback (MVF) increases the interlimb transfer but the exact mechanism is still a matter of debate. The aim of this study was to compare between a bimanual task (BM) and a MVF task, within functionally rather than geometrically defined cortical domains. Measure Projection Analysis (MPA) approach was applied to compare the dynamic oscillatory activity (event-related synchronization/desynchronization ERS/ERD) between and within domains. EEG was recorded in 14 healthy participants performing a BM and an MVF task with the right hand. The MPA was applied on fitted equivalent current dipoles based on independent components to define domains containing functionally similar areas. The measure of intradomain similarity was a "signed mutual information," a parameter based on the coherence. Domain analysis was performed for joint tasks (BM and MVF) and for each task separately. MVF created 9 functional domains while MB task had only 4 functionally distinctive domains, two over the left hemispheres and two bilateraly. For all domains identified for BM task alone, similar domains could be identified in MVF and joint tasks analysis. In addition MVF had domains related to motor planning on the right hemisphere and to self-recognition of action. For joint tasks analysis, seven domains were identified, with similar functions for the left and the right hand with exception of a domain covering BA32 (self-recognition of action) of the left hand only. In joint task domain analysis, the ERD/ERS showed a larger difference between domains than between tasks. All domains which involved the sensory cortex had a visible beta ERS at the onset of movement, and post movement beta ERS. The frequency of ERD varied between domains. Largest difference between tasks existed in domains responsible for the awareness of action. In conclusion, functionally distinctive domains have different ERD/ERS patterns, similar for both tasks. MVF activates contralateral hemisphere in similar manner to BM movements, while at the same time also activating the ipsilateral hemisphere. Significance: Following stroke cortical activation and interhemispheric inhibition from the contralesional side is reduced. MVF creates stronger ipsilateral activity than BM, which is highly relevant of neurorehabilitation of movements.

Utility of leucocyte antigens in distinguishing between bacterial and viral infection in children.

INTRODUCTION: Accurate diagnosis of bacterial and viral infection is very difficult. Unfortunately, there is still no quick and discriminative diagnostic test that would help clinicians in establishing the diagnosis and taking a decision on treatment. The aim of the study was to compare the expression of antigens on phagocytes, which are involved in the first defence line during bacterial and viral infections in children, as a potential tool to distinguish the etiology of the infection. MATERIAL AND METHODS: The expression of CD35, CD32, CD88, and MHC class I on phagocytes in 49 blood samples from children with high fever and suspected infection as well as 19 healthy children (control group) was assessed by flow cytometry. Thirty-three children were diagnosed with bacterial and 16 with viral infection. Expression of antigens was analysed on a FACSCanto II flow cytometer according to mean fluorescence intensity (MFI) and antibody binding cites (ABC). RESULTS: Significant differences were observed for the following: CD32, CD35, CD88, and MHCI on granulocytes; CD32, CD35, CD88 on monocytes; and MHC-I ratio between groups were observed. The obtained results did not allow us to establish valuable score points for distinguishing between bacterial and viral infections. Classification and a regression tree using CD88 expression on granulocytes and CRP was developed. It enabled us to differentiate between the origin of infection with sensitivity and specificity of more than 90%. CONCLUSIONS: Utility of use of wide range antigens' expression on phagocytes for distinguishing between bacterial and viral infection in children has limited value. More adequate seems to be use of CD88 expression on granulocytes linked with CRP value.

A Method for the Measurement of Salivary Gland Function in Mice.

Patients with Sjögren's syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Drug-free remission: the goal of the future in management of patients with rheumatoid arthritis.

Management of patients with rheumatoid arthritis according to the "treat-to-target" strategy requires achievement of remission or low disease activity when remission cannot be achieved (mostly in patients with advanced disease). The assessment of remission and low disease activity is based on a number of definitions depending on the applied instruments which do not always correspond to one another. The role of biomarkers and imaging techniques (ultrasound and magnetic resonance imaging) in predicting the risk for disease relapse after achieving remission and tapering disease-modifying antirheumatic drugs treatment are presented. The concept of achieving the full control of inflammation including residua synovial inflammation and drug free-remission is discussed.

Prevalence of thyroid diseases and antithyroid antibodies in women with rheumatoid arthritis.

INTRODUCTION: Thyroid abnormal function and and/or autoimmune thyroid disease (ATD) are observed in 6% to 33.8% patients with rheumatoid arthritis (RA). OBJECTIVES: The aim of the study was to determine whether ATD is more prevalent in patients with RA compared to the control group involving age and sex-matched subjects without RA and whether these patients should be screened for thyroid disease. PATIENTS AND METHODS: In 100 patients with RA and 55 patients without RA (control group) hormonal thyroid function and antithyroid antibodies were assessed. RESULTS: ATD was more prevalent (16%) in patients with RA than in the control group (9%). The difference was no statistically significant. The RA patients with concomitant ATD had lower RA activity than non-ATD RA patients. Antithyreoglobulin (anti-TG) and antithyroid peroxidase (anti-TPO) antibodies were present in similar percentage of patients with RA (12% and 15%, respectively) and in the control group (9% and 18%, respectively). The most common thyroid dysfunction observed in both groups was subclinical hypothyroidism. An association between thyroid dysfunction and clinical symptoms suggestive of thyroid disease in RA patients was not demonstrated. In patients with RA low free triiodothyronine concentrations were significantly more common. CONCLUSIONS: A higher prevalence of ATD in female RA patients compared with controls indicates the need for screening not only of thyroid function, but also of the presence of anti-TPO antibodies as the ATD marker in RA patients. Their presence does not correlate with the occurrence of thyroid disorders in RA patients. Monitoring of thyroid function is of particular importance since as already shown the course of thyroid disease in RA patients is often asymptomatic.

Reactive arthritis.

Reactive arthritis (ReA) is a non-purulent joint inflammation that usually follows bacterial gastrointenstinal or urogenital infections. The classic presentation of ReA is characterized by an asymmetric arthritis usually in the lower limbs associated with urethritis, conjunctivitis and occurrence of other articular or extra-articular manifestations. ReA is classified as a type of seronegative spondyloarthopathy. Approximately 65-85% of patients with ReA are HLA-B27 positive. Regardless of the preceding infection, the clinical picture is similar, but management can differ according to the triggering infection. Treatment of Chlamydia-induced ReA should be started with antibiotics because of several mechanisms by which Chlamydia can cause persistent infection. The disease may have an acute or self-limited course, however some patients develop chronic arthritis.

[Elderly onset rheumatoid arthritis]. / Reumatoidalne zapalenie stawów o poczatku w wieku póznym.

Elderly-onset rheumatoid arthritis (EORA), defined as rheumatoid arthritis (RA) with the onset at the age > or =60 differs sligthy at presentation from younger-onset RA (YORA) by a more equal sex distribution, a higher frequency of an acute onset, more frequent involvement of large joints, especially of the shoulder, and higher disease activity. Longitudinal studies have shown greater disease activity, more severe radiographic damage and functional decline in patients with EORA than in those with YORA. These differences were only found in seropositive patients. In seronegative EORA patients a less severe course of the disease and a better outcome have been observed, with the clinical manifestations of polymyalgia rheumatica or remitting seronegative symmetrical synovitis with pitting edema. This article is a review of available data concerning differences between EORA and YORA.